The presence of bacterial pathogens is a well recognized cause of severe illness, so that there is an ever present need for the detection of such pathogens in both clinical specimens (i.e. blood, tissue, urine and other body extracts and fluids), agricultural specimens (such as food products) and environmental specimens (such as surfaces in food processing plants).
However, current tests for the detection of bacterial pathogens, such as in food, typically require a number of days to complete. During this period of time, between sampling and assay determination, fresh food and dairy products will enter the food chain and therefore be consumed by the public. If a test indicates the presence of pathogens, expensive product recalls may result, or, worse, before the test results are discovered an outbreak of sickness may occur.
As stated above, traditional methods to detect the presence of bacterial food pathogens require an extended period of time, basically due to the need for an enrichment/incubation period. This incubation/enrichment period is intended to allow for growth of these bacteria from a background of competing microorganisms and an increase in bacterial cell numbers to more readily aid in identification. In many cases a series of two or three separate incubations is needed to isolate the target bacteria. However, such enrichment steps can actually compromise test sensitivity by killing some of the cells sought to be measured.
In the standard FDA procedure for detection of Listeria in food products (Bacteriological Analytical Manual, 7th Ed., 1992; Chapter 10) 25 g or 25 ml of a food sample is mixed with 225 ml of enrichment broth. This sample in broth mixture is incubated for 2 days. At the end of days 1 and 2 a sample of the broth culture is streaked onto petri plates containing selective growth agar and these plates are incubated for an additional 1-2 days. Identification of Listeria colonies is done by eye. This identification, however, is subjective, and presumed colonies must be confirmed by additional tests, which require another 1-2 days. Because of growth of bacteria during the enrichment step, the number of colonies on the agar plates does not represent the number of bacteria in the original sample. This test can only detect the presence of bacteria, and cannot quantitate the numbers originally present in the sample.
More recent methods of bacteria detection in food products have attempted to reduce the time needed for enrichment or confirmation. Many of these procedures utilize antibodies. A typical procedure, exemplified by the Listeria-Tek and Salmonella-Tek assays (Organon Technica Corp.), is a two site assay. That is, one antibody is immobilized in a microtiter well and acts to capture the target bacteria. This allows for separation of the target bacteria from the sample. A second antibody labelled with an enzyme is used to detect the captured bacteria. Theoretically, such an assay could be used to detect bacteria directly in a food sample. The actual sensitivity limit of these assays, however, makes it necessary to culture the target bacteria from the food sample. Because of the need for enrichment, the assay still requires 24-48 hours, even though the confirmation step is reduced to 1-2 hours. Enrichment also makes quantitation impossible.
Another alternative method for Listeria ,detection is immunomagnetic isolation. In this procedure, antibodies to the bacteria of interest are immobilized on magnetic beads. The beads, with attached antibodies, interact with the target organisms, which can be separated from other sample material and microorganisms in a magnetic field. This procedure is intended to reduce or eliminate the 24-48 hours enrichment period. Production and use of magnetic beads have been described in U.S. Pat. Nos. 3,970,518 (Giaever), 4,230,685 (Senyi and Widder), 4,677,055 (Dodin et al.), and 4,695,393 (Whitehead et al.). Immunomagnetic beads have been used to isolate Salmonella (Vermunt et al., J. Appl. Bact. 72, 112, 1992), Staphylococcus aureus (Johne et al., J. Clin. Microbiol. 27, 1631, 1989) and Listeria (Skjerve et al., Appl. Env. Microbiol. 56, 3478, 1990) from foods, and Escherichia coli from fecal samples (Lund et al., J. Clin. Microbiol. 29, 2259, 1991). In all of these examples, immunomagnetic capture is very inefficient at low numbers of bacteria. At the low levels of bacteria significant in food microbiology (&lt;100 bacteria per gram), these methods cannot be used without enrichment. Interference by non-target organisms sometimes occurs, requiring the addition of selective enrichment, or a confirmation step, or both, to make a complete assay.
In summary, these existing "rapid" immunoassay procedures for bacteria detection in food samples all require at least one dilution of sample into growth medium, followed by an enrichment period, then an assay procedure which only utilizes a fraction of this final culture. The actual assay sample thus only corresponds to a small fraction of the original food sample. The bacterial culture step, or steps,, must therefore overcome this dilution factor, adding to the amount of needed culture time. The use of the enrichment step also makes quantification of bacteria impossible. In addition, the utilized enrichment steps may kill the bacteria sought to be identified, producing a high false negative rate.
The method of the present invention overcomes the low capture efficiency characteristic of all previous immunomagnetic procedures. Immunomagnetic capture of bacteria is a complex process, and several parameters are important in successful capture. Good recovery of bacteria from food samples requires strong interaction between the bacteria and the immunomagnetic beads. The higher efficiency of the assay of the present invention results from improvements in both particle design and the method used for magnetic isolation.
A combination of bead porosity, bead size, attachment method, and long capture time accounts for the improved performance. The improvement in capture efficiency is such that bacteria can be isolated directly from food samples, without enrichment, at levels of less than 10 bacteria per gram of food. Because enrichment is avoided, the method allows quantitation of the number of bacteria present. In addition, the bacterial colonies formed are individually confirmed as target bacteria by an immunochemical confirmation step.